Project Summary/Abstract: Recent advances in tumor immunology have led to novel immune-based therapeutics, extending patient lives and in some cases resulting in permanent cures. These drugs block inhibitory receptors on cytotoxic CD8 T cells, leading to enhanced activation and tumor destruction. However, many tumors lack relevant antigens, or lose MHC I molecules, which are needed for displaying tumor antigens to CD8 T cells. Therefore, knowledge of how the immune system can be mobilized to kill MHC-deficient, CD8 T cell-resistant tumors is needed to address this potential escape mechanism and design next generation immunotherapies. Recently, intratumoral injection (IT) of STING-activating cyclic-di-nucleotides (CDNs) was shown to trigger CD8-dependent tumor rejection. Interestingly, my research shows that CDN injections also trigger rejection of MHC-deficient tumors in a CD8 T cell-independent process. Preliminary data show that Natural Killer (NK) and CD4 T cells mediate tumor rejection. Thus, CDNs may be used to uncover novel mechanisms of immune activation that lead to tumor destruction. This proposal aims to understand the CDN-induced rejection of MHC- deficient tumors by NK and CD4 T cells, with the ultimate aim of amplifying these forms of tumor rejection. In Aim 1, the mechanism of NK-dependent rejection will be established. The mechanism of action of the type-I interferon receptor (IFNAR) in CDN-induced NK activation will be determined using neutralizing antibodies and cell-specific knockout mice. The roles of IFNAR-dependent NK-activating cytokines will also be addressed. NK-mediated antitumor effector mechanisms will be assessed with Prf1-/-, IFN?-/-, and T cell- deficient Rag2-/- mice. Tumor cells will be engineered using CRISPR-Cas9 to lack the IFN?R, Fas, and other known NK-activating ligands and death receptors, to determine their roles and sites of action. In Aim 2, the mechanism of CD4-dependent rejection will be established. The role of IFNAR in priming CD4 T cells will be addressed using tumor-specific peptide stimulation in vitro, and cell-specific knockout mice in vivo. Antitumor CD4 T cell effector mechanisms will be addressed using Prf1-/-, IFN?-/-, and NK-deficient NKDTA mice, depleting and neutralizing antibodies, and genetically engineered tumor cells. In Aim 3, cooperative mechanisms between NK and CD4 T cells will be established. The role of CD4 T cells in activating NK cells will be addressed using cell-depleting antibodies, add-back adoptive transfers, and NK activation assays. Roles of NK-activating molecules produced by CD4 T cells will be determined using ex vivo peptide stimulation in combination with specific neutralizing antibodies, followed by NK activation assays. The role of NK cells in activating and recruiting CD4 T cells to tumors will be assessed using cell depletion and adoptive transfer experiments followed by ex vivo CD4 peptide stimulation assays and flow cytometry of tumor cells ex vivo. This project aims to uncover how the immune system can be used to target MHC-deficient, CD8- resistant tumors with the conviction that the results will lead to the development of novel cancer therapies.